Production and purification of amino acids



Patented June 16, 1953 PRODUCTION AND PURIFICATION OF AMINO ACIDSHalbert C. White, Midland, Mich., assignor to The Dow Chemical Company,Midland, Mich., a corporation of Delaware No Drawing. ApplicationDecember 8, 1948, Serial No. 64,239

9 Claims.

This invention pertains to an improved method for the production andpurification of alphaamino monocarboxyllc acids. It pertains especiallyto certain new steps for separating alpha-amino acids, particularlywater-soluble alpha-amino acids, from water-soluble inorganic saltimpurities incident to manufacture of the amino acids.

It is known to prepare alpha-amino-monocarboxylic acids by reactingtogether, in an aqueous medium, an ammonium carbonate, a cyanide such ashydrogen cyanide or an alkali metal cyanide, and an aldehyde or ketoneto form a hy dantoin, separating the later, and hydrolyzing thehydantoin to form an alpha-amino acid or another hydantoin to itscorresponding alphai amino-monocarboxylic acid.

Furthermore, when this known method is applied under conditions such asto form an alphaamino-monocarboxylic acid, the latter is obtainedtogether with various organic impurities and water-soluble inorganicsalts such as alkali metal carbonates and chlorides, etc. Certain of theinorganic salts, particularly sodium chloride, are usually present asimpurities in agents used in the synthesis and other salts, e. g.,alkali metal carbonates, are formed in' the process. The proceduresheretofore employed for separation of the amino acids from theimpurities have difiered, depending upon the kind of amino acid beingprepared, and -in many instances have been inconvenient and costly.Separation of the water-soluble amino acids from the soluble inorganicsalt impurities has been particularly troublesome. Accordingly, althougha number of amino acids have been prepared on a laboratory scale inaccordance with this known scheme of synthesis, the latter has notheretofore been satisfactorily applicable for the manufacture of many ofthe amino acids which, at least theoret-- ically, may be prepared inaccordance with'such scheme. 7

In my I co-pending application, Serial No. 708,461, filed November 7,1946, now Patent No. 2,557,920, of which this application is acontinuation-in-part, it is disclosed that hydantoins having the generalformula:

wherein R and R each individually represents a member of the groupconsisting of hydrogen and monovalent organic radicals having a carbonatom thereof attached to the hydantoin nucleus, may be hydrolyzed toform a salt of an alphaamino-monocarboxylic acid in good yield byheating the hydantoin together with an aqueous solution of an alkalimetal hydroxide, or an alkali metal carbonate, under pressure in aclosed vessel at a reaction temperatureabove 130 C. and preferably inthe range of from 130 to 300 C. The hydantoins having the above-statedgeneral formula are those formed by a reaction between an inorganiccyanide, an ammonium carbonate, and an aldehyde or ketone. Thehydrolysis method of said co-pending application is applicablemonocarboxylic acid from any such hydantoin. The amino acid is formeddirectly as an aqueous solution of an alkali metal salt thereof, hencethe hydrolysis may conveniently be carried out either batchwise, or in acontinuous manner,

e. g., by passage of the reaction mixture under pressure through aheated tubular autoclave.

Although the hydrolysisv method of said copending application, SerialNo. 708,461, may be applied to form soluble salts of any of a widevariety of alpha-amino-monocarboxylic acids in good yield, separationand purification of the amino acids to remove the aforementionedimpurities incident to formation of the same has in many instances beendifficult. The procedures heretofore employed for isolation of thewatersoluble amino acids in a form substantially free of theaforementioned soluble inorganic salt impurities has been particularlydifiicult.

It is an object of this invention to provide an improved method for theproduction and purification of alpha-amino-monocarboxylic acids byreacting together an ammonium carbonate, an inorganic cyanide, and analdehyde or ketone to form a hydantoin, hydrolyzing the latter with anaqueous alkali metal base solution to form a solution of analkali metalsalt of the amino acid together with the aforementioned water-solubleinorganic salt impurities, and isolating the amino acid from the reactedmixture. Afurther object is to provide such a method, whereby theconvenin producing an alpha-amino is to provide certain new steps forthe recovery,

in purified form, of an alpha-amino-monocarboxylic acid from an aqueoussolution comprising an alkali metal salt of the same together withwater-soluble inorganic salt impurties incident to preparation of theamino acid. Other objects will be apparent from the followingdescription of the invention.

I have found that an alpha-amino-monocarboxylic acid may readily beseparated in purified form from an aqueous solution containing the sametogether with the aforementioned usual organic and inorganic saltimpurities by evaporating, ordistilling, water from the solution toobtain a residual solid mass, or aqueous slurry, of the salts; adding alower alcohol, and Water, if necessary, to the residue in proportionssuch that the alcohol corresponds to from 60 to 98, preferably from 80to 90, per cent of the combined weight of the same and the waterpresent, whereby the alkali metal salt of the amino acid is selectivelydissolved, or extracted, from the slurry by the aqueous alcohol solutionto leave the inorganic salt impurities in the residue; separating theresultant alcoholic solution and neutralizing it by addition of anorganic carboxylic acid which, when converted to the sodium or potassiumsalt thereof, isreadily soluble, e. g., to form an at least 2 weight percent solution thereof, in an aqueous methyl alcohol solution of 90weight per cent concentration, whereby the free amino acid isprecipitated from the alcoholic solution; and separating the amino acidfrom the liquor. In this way, any of the alpha-aminomonocarboxylic acidsformed by hydrolysis, with an aqueous alkali metal base solution, ofhydantoins having the aforementioned general formula, may readily berecovered from the hydrolysis liquor in good yield and in a form of highpurity.

The above new procedure for the isolation of an amino acid from analkaline hydrolysis mixture containing the same in impure form is basedon certain other discoveries, viz. that alkali metal salts of thealpha-amino-monocarboxylic acids are readily and rapidly dissolved by anaqueous alcohol solution of the above-stated concentration, whereasinorganic salt impurities incident to preparation of the amino acids areinsoluble, or at most only sparingly soluble, in such alcoholicsolution; and that the free amino acids are sparingly soluble in theaqueous alcohol solution, whereas the organic impurities incident topreparation of the amino acid are soluble in the solution.

For effective purification of the amino acid product it is importantthat the aqueous alcohol solution contain from 60 to 98 per cent of thealcohol, based, on the combined weight of the alcohol and water in themixture. A ueous alcohol solutions of lower concentration usuallydissolve an obiectionable amount of the inorganic salt im uritiestogether w th the alkali metal salt of the amino acid product. Analcohol of greater than 98 per cent concentration frequently convertsthe alkali metal carbonate, or alkali metal bicarbonate. im urity to aslimy or gelatinous material, presumably by forming a double compoundtherewith. The presence of such limy or gelatinous material interfereswith precipitation of the inorganic salt impurities and also withfiltration of the mixture by plugging the filter. In practice theaqueous alcohol employed for selective extraction of the alkali metalsalt of the amino acid is preferably of from to concentration by weight.It is used in amount sufficient to dissolve at least 80 per cent ofthesalt of the amino acid without dissolving all of the salts present.Usually between 5 and 30, preferably between 8 and 15, parts by weightof alcohol, on an anhydrous basis, is used per part of amino acid(expressed as free amino acid) in the residue. By carrying theextraction out in a Soxhlet extractor so as repeatedly to reuse the samealcohol, the latter may be employed in proportions considerably lowerthan just stated.

I have further found that the above-mentioned steps for recovery andpurification ofan amino acid are efiective in separating such productfrom the impurities normally present together with the intermediatehydantoin product, and that the conventional steps of separating thehydantoin from the aqueous solution in which it is formed, prior tohydrolyzing the hydantoin, may advantageously be omitted. According tothe present invention, a crude aqueous reaction mixture comprising ahydantoin and its usual impurities may be treated directly with anaqueous alkali metal base and the hydantoin be hydro- I lyzed, to obtaina mixture from which an amino acid of high purity may be separated. bythepurification steps of the invention. Omission of the usual steps forseparation and purification of the hydantoin intermediate product is ofimportance not only because of the reduction in the number of stepsinvolved, but also because a considerable portion of the hydantoinusually is lost or destroyed during such steps for isolation of the samefrom the aqueous mixture in which it is formed, i. e., omission of saidsteps usually results in an improved yield of the amino acid product. Asan illustration, it may be mentioned that it has not heretofore beenpossible to isolate the compound, hydantoin, from the crude aqueousmixture resulting from a reaction between formaldehyde, sodium cyanideand ammonium carbonate. However, I have found that such crude reactionmixture may be treated directly with an alkali metal base andbe heatedunder pressure at C. or above to form a hydrolysis mixture from whichglycine of high purity may be recovered in a substantial yield by thepurification procedure of the invention.

Inasmuch as procedures for reacting together, in an aqueous medium, aninorganic cyanide, ammonium carbonate or bicarbonate, and an aldehyde orketone to form an aqueous solution of a hydantoin having theaforementioned general formula, and agents which may be employed in thereaction to form such hydantoins, are known, they need not be describedin detail. However, it may be mentioned that the identity of thehydantoin is dependent upon the kind of aldehyde or ketone employed as areactant and that the reaction results in a crude aqueous solution of ahydantoin together with organic and inorganic salt impurities.Heretofore, it has been usual practice to separate and purify thehydantoin prior to hydrolyzing the same. By such reaction,S-methyl-hydantoin may be formed using acetaldehvde as a reactant,S-ethyl-hydantoin may be formed from propionaldehyde, 5-propylhydantoinfrom butyraldehyde, 5-isopropyl-hydantoin from isobutyraldehyde,5-butyl-hydantoin from valeraldehyde, 5-isobutyl-hydantoin fromisovaleraldehyde, 5-phenyl-hydantoin from benzaldehyde, 5 (betamethylmercaptoethyl) hydantoin from gamma-methylmercaptopropionaldehyde;5,5-dimethyl-hydantoin from acetone, and 5,5-diethyl-hydantoin fromdiethyl ketone, etc.

According to the present invention, a hydantoin, or preferably one ofthe above-mentioned crude aqueous reaction mixtures having an impurehydantoin dissolved therein, is treated with an alkali metal base suchas sodium hydroxide, potassium hydroxide, sodium carbonate, or potassiumcarbonate, etc., in amount greater than that theoretically required forformation of an alkali metal salt of the alpha-amino-monocarboxylic acidobtainable by hydrolysis of the hydantoin. Two or more, preferably from2 to 4, molecular equivalents of the alkali are usually employed permole of the hydantoin, but the alkali may be used in a somewhat smallerproportion or in as great a proportion as desired. The aqueous alkalisolution, in which the hydantoin is thus dissolved, may be of anydesired concentration, but usually contains from 2 to per cent by weightof the alkali. When treating an aqueous reaction mixture in which ahydantoin was formed, the alkali may be added as a solid or as aconcentrated aqueous alkali solution.

The mixture is heated in a closed bomb or autoclave at temperaturesabove 130 C., e. g. from 130 to 300 C. or higher and preferably between140 and 250 (3., until the hydrolysis of the hydantoin is nearlycomplete. Since the hydrolysis to form an amino-monocarboxylic acid saltresults in formation of one molecular equivalent of ammonia per mole ofthe hydantoin consumed, the extent of the reaction may be determined atany time by withdrawing an aliquot portion of the reaction mixture anddetermining the ammonia content thereof. When operating at a temperatureof 150 0., the reaction usually is nearly complete in one-half hour. Athigher temperatures it may be completed in a shorter time, e. g., insome instances in one minute. The mixture is advantageously cooled, e.g., to less than 130 C., as promptly as possible after completion of thehydrolysis reaction, since longer heating may result in decomposition ofa portion of the product. When carrying the reaction out in a continuousmanner, e. g., by passage of the mixture under pressure through atubular autoclave and fromthe latter through a relief-valve, cooling maybe efiected by evaporation of water from the mixture as it flows fromthe autoclave to a zone of lower pressure. In this way, heat stored inthe hydrolysis liquor may be used to concentrate the product.

After withdrawal from the bomb or autoclave, the hydrolysis mixture isconcentrated by evaporation of water therefrom to obtain a solid mass,or preferably an aqueous slurry, of salts as a residue. Such evaporationmay be carried out at atmospheric or at subatmospheric pressure, or evenat a moderately increased pressure, e. g., up to pounds per square inchgauge if desired. Evaporation of the hydrolysis mixture may be precededby intervenin steps of treating the hydrolysis liquor with adecolorizing agent, such as activated charcoal or a bleaching earth, andfiltering, or such decolorizing treatment may be omitted or be deferredto a later stage in the process.

The residue is treated with a lower alcohol in amount corresponding tofrom 60 to 98, preferably from to 90, per cent of the combined weight ofthe same and the water present. Any lower alcohol, or ether-alcohol,having not more than 5 carbon atoms in the molecule and free of otherfunctional groups may be used for the pur-' pose. Examples of such loweralcoholic compounds are methanol, ethanol, propanol, ethylene glycol,propylene glycol, methoxymethanol, ethoxymethanol, ethoxyethanol, .andmethoxypropanol, (i. e., the monomethyl ether of propylene glycol), etc.Methanol is preferably employed.

The inorganic salts are substantially insoluble in the added alcohol,but the alkali metal salt of the alpha-amino-monocarboxylic acid productdissolves, together with any organic impurities, in the aqueous alcohol.The inorganic salts are removed, e. g., by filtration or decantation.Thereafter, the alcoholic liquor is neutralized with a carboxylic acidwhich is of strength greater than that of the amino acid and which formsan alkali metal salt that is soluble in the alcoholic medium. A varietyof carboxylic acids are known which are suitable for the purpose.Examples of such acids are formic acid, acetic acid, propionic acid,oxalic acid, succinic acid, and benzoic acid, etc. I

By adding the organic acid to the alcoholic solution in amount such asto bring the mixture to a pH value of from 4 to 8, advantageously from 5to 7, and preferably about 6, the amino acid is liberated from itsalkali metal salt and precipitated, leaving the organic impurities andan alkali metal salt of the organic acid neutralizing agent in solution.The precipitate is removed, e. g., by filtration, washed with a loweralcohol, or other volatile organic liquid non-solvent for the aminoacid, and dried. It is thereby obtained in a form of high-purity. Whennecessary, the amino acid may be decolorized in usual ways, e. g., byforming a solution thereof, treating the solution with a decolorizingagent such as activated carbon or a bleaching earth, filtering, andrecovering the amino acid from the filtrate. As hereinbefore indicated,the decolorization may be accomplished while the amino acid is dissolvedas a salt in the hydrolysis mixture in which it is formed.Alternatively, it maybe accomplished using the aqueous alcohol solutionof such salt, or it may be accomplished after isolation of the aminoacid product. In some instances, decolorization is not required and thedecolorizing steps may be omitted.

The following examples describe certain ways in which the invention hasbeen practiced, but are not to be construed as limiting its scope.

Example 1 Approximately 58 pounds (0.8 mole) of isobutyraldehyde wasadded gradually, over a period of 40 minutes, to a solution of pounds ofammonium bicarbonate and 42 pounds of sodium cyanide in 80 gallons ofwater. The mixture was then heated at a temperature of 80 C. for 2.5hours, whereby 5-isopropylhydantoin was formed. Thereafter, the mixturewas boiled at atmospheric pressure, to vaporize water and unreactedammonium bicarbonate therefrom, until the temperature of the residualmixture had increased to 102 C. Approximately 10.5 gallons of an aqueoussodium hydroxide solution of 50 per cent concentration was then added.The resultant solution was passed through a tubular autoclave, heated toabout C., at a pressure greater than the autogenous pressure and at arate of flow such that the contact time, i. e., the time for passage ofan infinitesimal portion 01' the mixture through the autoclave, wasabout 44 minutes. After discharge from the autoclave, the reactionliquor was evaporated by boiling at atmospheric pressure until itstemperature had risen to 115 C. and the residue was an aqueous slurry ofsalts. The slurry was permitted to cool somewhat and 55 gallons ofaqueous methanol, containing about 90 per cent by weight of methanol,was added. A considerable portion ofthe solids present in the slurrywere thereby dissolved, but the inorganic salts present in the mixturewere substantially insoluble and settled in solid form. The mixture wascooled to 40 C. and filtered to remove the solids, which consistedprincipally of sodium carbonate and sodium bicarbonate together withother salts such as sodium chloride. The filtrate was brought to a pHvalue of by treatment with acetic acid, whereby the amino acid product,i. e., valine, was precipitated. The mixture was cooled to C. andfiltered to remove the valine and the latter was washed with methanoland dried. There was obtained 46 pounds of valine, the yield being 49per cent of theoretical based on the isobutyraldehyde starting material.

Example 2 An aqueous solution of crude S-ethylhydantoin was prepared byheating a mixture of 93 pounds (1.6 moles) of propionaldehyde, 34 poundsof sodium cyanide, 274 pounds of ammonium bicarbonate and 160 gallons ofwater at a tempera- 1' ture of 80 C. for 2.5 hours. Thereafter, themixture was boiled to vaporize water and unreacted ammonium bicarbonatetherefrom until the temperature had increased to 102 C. Approximatelywas added, whereby a large part, but not all,

of the solid material in the slurry was dissolved. The alcoholic extractwas separated from the undissolved salts. The residue was extracted withtwo further 15 gallon portions of methanol and the several alcoholicextracts were combined. The alccholicliquor was then neutralized withacetic acid, as in Example 1, whereby the product, dl-alpha-aminobutyricacid, was precipitated. The product was separated by centrifuging themixture, washed with methanol, and dried. There was obtained 106 poundsof white crystalline dl-alpha-amino-butyric acid having a melting pointof 270-272 C. The yield of crystalline alpha-aminobutyric acid was 64per cent of theoretical, based on the propionaldehyde starting material.

Example 3 A mixture of 70 pounds (1.6 moles) of acetaldehyde, 234 poundsof ammonium bicarbonate, 84 pounds of sodium cyanide, and 180 gallons ofwater washeated to 80 C. for 2.5 hours, whereby 5-methylhydantoin wasformed. Water and unreacted ammonium bicarbonate were vaporized byboiling the mixture until the temperature ior'boilin'g had risen'to 102C. Approximately gallons of an aqueous sodium hydroxide solution of 50per cent concentration was added. The mixturewas passed under pressurethrough a tubular autoclave heated to 170 C. at a rate of flowcorresponding'to a contact time of between 40 and minutes. The effluentliquor was evaporatecl until the boiling temperature had risen to about125 C. and the residual material consisted of an aqueous salt slurry.The residue was extracted first with 110 gallons of methanol andthereafter with two 15 gallon portions of methanol and the extracts werecombined. Inorganic salts such as sodium carbonate, sodium bicarbonateand sodium chloride, remained in the residue from the extraction. Thealcoholic liquor was neutralized with acetic acid, Whereby alanine wasprecipitated. The precipitate was removed by centrifuging the mixture,washed with methanol and dried. There was obtained 100 pounds ofcrystalline dl-alanine of high purity. The yield was per cent oftheoretical, based on the acetaldehyde starting material. 1

- Ezcample 4 Approximately 16.5 grams of an aqueous formaldehydesolution, containing 40 per cent by weight or 0.2 gram mole offormaldehyde, was added to a solution of 34.5 grams (0.437 mole) ofammonium bicarbonate, 10.5 grams (0.264

mole) 01 sodium cyanide, and 160 cc. of water. The mixture was heated atC. for 3 hours and thereafter was evaporated to about 60 per cent of itsinitial volume by boiling the same at atmospheric pressure.Approximately 42 cc. of a 30 per cent aqueous sodium hydroxide solutionwas added and the mixture was heated in a closed iron bomb at 175 C. for30 minutes. The bomb was then cooled and the mixture removed. The

mixture was then decolorized with activated carbon, filtered, and thefiltrate was evaporated nearly to dryness. The residual salt mixture,which comprised hydrates of sodium carbonate and sodium bicarbonate, wasextracted with about cc. of methanol, whereby the sodium salt of theglycine product was dissolved leaving the inorganic salts in theresidue. Glycine was precipitated from the extract by bringing thelatter to a pH value of 5 with acetic acid. The glycine was separated byfiltration, washed with methanol, and dried. There was obtained 4 grams,or 26.5 per cent of the theoretical yield, of substantially pureglycine.

Example 5 One hundred grams of crude dl-alanine, containing 34 per centby weight of ammonium chloride, was dissolved in 300 cc. of water and112 grams of a 50 per cent aqueous sodium hydroxide solution was added.The solution was evaporated by boiling until a salt slurry, containingabout 60 grams of water, remained. The slurry was treated with 400 cc.of methanol and the mixture filtered, leaving inorganic salts, e. g.sodium chloride, as the residue. The filtrate was neutralized withacetic acid to a final pH value of between 5 and 6, whereby alanine wasprecipitated. The precipitate was removed by filtration, washed withmethanol and dried. There was obtained 52 grams of substantially purealanine having an ash value of only 0.3 per cent.

Example 6 An aqueous solution of crude tryptophane, obtained byhydrolyzing 0.1 gram mole of 5-(3- indolylmethyl) hydantoin with aqueoussodium hydroxide, was divided into two equal portions. One portion wastrated with activated carbon, filtered, and the filtrate was neutralizeddirectly with acetic acid, and the tryptophane thus precipitated wasfiltered from the mixture, washed with methanol and dried. There wasobtained 7.5 grams of tryptophane of good purity, but having a tancolor. The other portion of the hydrolysis mixture was evaporated byboiling until an aqueous salt slurry remained. The slurry was extractedwith two 100 cc. portions of methanol, and. the combined extract wasdecolorized with activated carbon, filtered ,and neutralized withTacetic acid to precipitate dl-tryptophane. The latter was filtered fromthe mixture, washed with methanol and dried. There was obtained 7 gramsof white tryptophane of high purity.

Other modes of applying the principle of the invention may be employedinstead of those explained, change being made as regards the methodherein disclosed, provided the step or steps stated by any of thefollowing claims or the equivalent of such stated step or steps beemployed. 3

I therefore particularly point out and distinctly claim as myinvention: 1. In a method wherein an aqueous solution of a hydantoin,having the general formula:

in which R and R each individually represents a member of the groupconsisting of hydrogen and monovalent organic radicals having a carbonatom thereof attached to the hydantoin nucleus, is treated with analkali metal base in amount corresponding to at least twice themolecular equivalent of the hydantoin, and the mixture is heated tohydrolyze the hydantoin and thus form an aqueous solution of an alkalimetal salt of an alpha-amino-carboxylic acid, the steps of evaporatingwater from the hydrolysismixture until dissolved salts are crystallizedtherefrom, extracting the alkali metal salt of the alpha-amino car,-boxylic acid from the residue with an aqueous alcohol solution,containing a lower alcohol in a concentration of from 60 to 98 per centby weight, to leave inorganic salts in the residue, precipitating thealpha-amino-monocarboxylic acid from the extract by neutralizing thelatter with a stronger carboxylic acid to leave the resultant alkalimetal salt of the stronger carboxylic acid in solution, and separatingthe crystalline alphaamino-monocarboxylic acid product.

2. A method, according to claim 1, wherein a soluble salt of awater-soluble alpha-aminomonocarboxylic acid is formed by thehydrolysis, and wherein the alcohol of the extractant is a saturatedmonohydric alcohol containing not more than three carbon atoms in themolecule.

3. A method, according to claim 1, wherein a soluble salt 'of awater-soluble lalpha-amino- 1 9 monocarboxylic acid is formed by thehydrolysis, the alcohol of the extractant is methanol, and thealpha-amino-monocarboxylic acid product is precipitated from thealcoholic extract by neutralizing the latter with acetic acid. I

4. In a method for separation of an alphaamino-monocarboxylic acid froman aqueous concentration of from 60 to 98 per cent by weight,

to leave the inorganic salt impurity in the residue, precipitating thealpha-amino-monocarboxylic acid from the extract by neutralizing thelatter with a stronger carboxylic acid which is dissolved, as an alkalimetal salt thereof, in the aqueous alcohol, and separating thecrystalline alphaamino-monocarboxylic acid product. i

5. A method, according to claim 4, wherein thealpha-amino-monocarboxylic acid is one which is soluble in water and thealcohol of the extractant is a satuarted alcohol having not more thanfive carbon atoms in the molecule.

6. A method, according to claim 4, wherein thealpha-amino-monocarboxylic acid is one'which is soluble in water, thealcohol of the extractant is methanol, and the carboxylic acidneutralizing agent isacetic acid.

7. A method, according to claim 6, wherein thealpha-amino-monocarboxylic acid is alanine.

' 8. A method, according to claim 6, wherein thealpha-amino-monocarboxylic acid is isoleucine.

9. A method, according to claim 6, wherein thealpha-amino-monocarboxylic acid is alphaamino-butyric acid.

HALBERT C. WHITE.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 427,565 Gnehm May 13, 1890' 1,602,958 Woo Oct. 12, 19261,634,222 Tressler June 25, 1927 2,109,929 Rigby Mar. 1, 1938 2,225,155Cheronis Dec. 17, 1940- 2,384,817 Chitwood Sept. 18, 1945 2,446,192Pfister et a1. Aug. 3, 1948 2,456,742 Shabica Dec. 21,1948 2,480,644Goldsmith, et a1. Aug. 30 194 FOREIGN PATENTS Number Country Date594,275 Germany Mar. '14, 1934 257,403 Switzerland Apr. 16, 1949 OTHERREFERENCES Livak et al., J. Am. Chem. Soc., vol. 67, pp. 2218-2220(1945).

1. IN A METHOD WHEREIN AN AQUEOUS SOLUTION OF A HYDANTOIN, HAVING THE GENERAL FORMULA: 